Cytotoxic nucleotides for targeted therapeutics

ABSTRACT

The present invention provides a method of generating a nucleic acid, which specifically binds to an extracellular surface protein expressed by a cell of interest, and which nucleic acid comprises a compound of interest to be delivered to the cell of interest.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 60/771,232, filed Feb. 8, 2006,the disclosure of which is incorporated herein by reference in itsentirety.

GOVERNMENT SUPPORT

This invention was made with government support under grants from theDepartment of Defense and the National Institutes of Health. Thegovernment has certain rights to this invention.

FIELD OF INVENTION

The present invention concerns chemotherapeutic molecules andcompositions thereof, and methods of use thereof for the treatment ofcancer.

BACKGROUND OF THE INVENTION

Cancer is the second-leading cause of death in the United States and isa serious public health concern. The current generation of cytotoxicchemotherapeutic agents used for the treatment of cancer is not curativefor a majority of patients. For many cancer patients, the use ofchemotherapy extends patient-life by only a few months and often resultsin serious side effects that reduce the quality of life.

Anticancer drugs that are utilized for cancer chemotherapy includecytotoxic nucleoside analogs (Pratt et al., “Antimetabolites” in TheAnticancer Drugs, 2^(nd) ed. Oxford University Press, New York. pp.69-107 (1994)), such as analogs of the four nucleotides that are theprincipal components of DNA. Examples of cytotoxic analogs include thefluoropyrimidines (FPs) such as 5FU, which are analogs of dU, theprecursor for dT, the arabinosyl nucleotides AraC and AraA, which areanalogs of dC and dA, respectively, dFdC (gemcitabine), which is ananalog of dC, and 6-mercaptopurine, which is an analog of dI, theprecursor of dG.

The current paradigm in chemical drug development involves restrictionson the molecular weight and the charge of candidate drugs. The rationalefor these restrictions is that drugs must have high bioavailability andmust enter cells either by passive diffusion or by well-characterizedmolecular transport processes. Thus, while the activated forms ofnucleoside analogs are typically 5′-O-mono-, di-, or tri-phosphates,cytotoxic nucleoside analogs are either administered as the nucleobase,if the active form of the drug is the 2′-deoxyribonucleotide (e.g. 5FUas a precursor for FdUMP), or as the nucleoside, if the activated formof the drug has a non-native sugar (e.g. AraC as a precursor forAraCTP). However, 2′-deoxyribonucleosides are generally ineffective asdrugs because of the facile cleavage of the glycosidic bond.

The requirement for intracellular metabolic activation of these drugsdecreases their effectiveness for at least two reasons: 1) cancer cellscan become resistant to the drug by down-regulating the expression ofcellular enzymes that are required for metabolic activation; and 2)competing metabolic processes may divert the drug to undesirableproducts in the cell. For example, 5FU is administered as a precursor toFdUMP but is also metabolized to FUTP and incorporated into RNA,resulting in toxicity towards cells of the gasterointestinal tract(Pritchard et al., Proc. Natl. Acad. Sci. USA 94: 1795-1799 (1997)).

The principal cause for the ineffectiveness of cytotoxicchemotherapeutic drugs in current use (e.g., 5-fluorouracil (5FU)) is afailure of the activated form of the drug to accumulate in cancer cellsat sufficient concentrations to cause cancer cell death (Longley et al.,Nature Cancer 3: 330-338 (2003)). Malignant cells that survive drugtreatment become drug-resistant, and refractory to further chemotherapy.

For example, despite great efforts utilizing conventional chemotherapystrategies, metastatic prostate cancer (PC) currently remains incurableand an inevitably fatal disease. This is largely due to the fact thatchemotherapeutic drugs are not effective at killing tumors in late-stagePC because PC cells do not accumulate drugs at sufficient concentrationsto cause cell death. The ability to deliver activated cytotoxic drugsspecifically to PC cells in vivo is likely to result in a reduction ofthe mortality rate (currently 30,000 per year) for patients withadvanced PC (American Cancer Society Facts and Figures 2004,http:/www/cancer.org).

Targeted delivery of cytotoxic drugs is expected to decrease themorbidity associated with cancer chemotherapy. Thus, there is a greatneed for innovative approaches to improve the long-term survival ofpatients suffering with this disease.

SUMMARY OF THE INVENTION

Provided herein are methods of generating a nucleic acid of interest,which nucleic acid specifically binds to an extracellular surfaceprotein expressed by a cell of interest, and which nucleic acid iscapable of being internalized by said cell of interest, said methodcomprising the steps of: (a) combining a first pool comprising differentnucleic acids with said extracellular surface protein; (b) selecting afirst subpopulation of nucleic acids from said first pool, said firstsubpopulation comprising at least one nucleic acid that specificallybinds to said extracellular surface protein; (c) amplifying said atleast one nucleic acid of said first subpopulation; and (d) selecting asecond subpopulation comprising at least one nucleic acid species fromsaid first subpopulation which is internalized by said cell of interest.Cells of interest may be, e.g., cancer cells, microbial cells, parasiticcells, etc. The nucleic acid may comprise a compound of interest, suchas an active compound (e.g., cytotoxic nucleotides) and/or a detectablegroup.

Also provided are methods of generating a nucleic acid of interest,which nucleic acid specifically binds to an extracellular surfaceprotein expressed by a cell of interest, which nucleic acid is capableof being internalized by said cell of interest, and which nucleic acidcomprises one or more compounds of interest to be delivered to said cellof interest, said method comprising the steps of: (a) combining a firstpool comprising different nucleic acids with said extracellular surfaceprotein; (b) selecting a first subpopulation of nucleic acids from saidfirst pool, said first subpopulation comprising at least one nucleicacid that specifically binds to said extracellular surface protein; (c)amplifying said at least one nucleic acid of said first subpopulation;(d) selecting a second subpopulation comprising at least one nucleicacid species from said first subpopulation which is internalized by saidcell of interest; (e) determining a first chemical structure of anucleic acid from said second population; (f) determining a secondchemical structure of a nucleic acid which is an analog of said nucleicacid from said second population, said analog comprising one or more ofsaid compounds of interest; (g) analyzing the folding properties of saidfirst chemical structure; (h) analyzing the folding properties of saidsecond chemical structure; (i) comparing the folding properties of saidfirst chemical structure with that of said second chemical structure;and (j) generating a nucleic acid of interest based upon said comparing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of FdUMP[5], the linear homopolymer of FdUMPnucleotides of length 5, and the chemical structures for 5-fluorouracil(5FU), 5-fluoro-2′-deoxyuridine (FdU), and5-fluoro-2′-deoxyuridine-5′-O-monophosphate (FdUMP).

FIG. 2. Diagram of a selection of a first pool of nucleotides thatselectively bind to a differentially expressed extracellular surfaceprotein bound to an affinity matrix. The pool comprises nucleotideshaving two constant regions (A and C) flanking a random sequence region(B).

FIG. 3. The lowest energy (A) and second lowest energy (B) secondarystructures of the A10-3 aptamer (SEQ ID NO:1) as predicted from theprimary sequence using mFOLD. (C) Proposed secondary structure for amodified aptamer generated from the A10-3 aptamer sequence (SEQ IDNO:3).

FIG. 4. Circular dichroism spectra and UV thermal melt data of themodified A10-3 aptamer (A) and (C), respectively, and the parent A10-3aptamer (B) and (D), respectively.

FIG. 5. Fluorescence and phase contrast images of LNCaP and PC3 cellsfollowing exposure to modified A10-3 aptamers conjugated with the FAMdye. The fluorescence images of LNCaP cells following a 15 min or 45 minexposure to the modified aptamer are shown in FIGS. 5B and 5D,respectively. The corresponding phase contrast images are shown in FIGS.5A and 5C. FIGS. 5E and 5F, respectively, show the phase contrast andfluorescence microscopy images of PC3 cells following 30 min exposure tothe modified aptamer.

FIG. 6. Cytotoxicity of a parent, unmodified, RNA aptamer (A) towardLNCaP prostate cancer cells as compared to the dose-dependentcytotoxicity of the RNA aptamer modified to contain a FdUMP[9] tail (B).

FIG. 7. Graphs of the percent viable cells following a 40 minuteexposure to the modified aptamer and 48 hour incubation in drug freemedium. (A) LNCaP cells; (B) PC3 cells.

FIG. 8. Bar graphs indicating the percent viable cells following 1 hourtreatment with A10-3:FdU[9]dC (solid bars) or A10-3:T[10] (hatchedbars). LNCaP (A) and PC3 (B) cells were exposed to the modified aptamersfor one hour.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all of thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound, dose, time,temperature, and the like, is meant to encompass variations of 20%, 10%,5%, 1%, 0.5%, or even 0.1% of the specified amount. Also, as usedherein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The disclosures of all Patent references cited herein are incorporatedherein by reference in their entirety.

“Compound of interest” as used herein includes, but is not limited to,detectable compounds and active compounds.

“Detectable compounds” as used herein include, but are not limited to,radiolabels (e.g., ³⁵S, ¹²⁵I, ³²P, ³H, ¹⁴C, ¹³¹I), enzyme labels (e.g.,horseradish peroxidase, alkaline phosphatase), gold beads,chemiluminescence labels, ligands (e.g., biotin, digoxin) and/orfluorescence labels (e.g., rhodamine, phycoerythrin, fluorescein), afluorescent protein including, but not limited to, green fluorescentprotein or one of its many modified forms, a nucleic acid segment inaccordance with known techniques, and energy absorbing and energyemitting agents.

“Active compound” as used herein includes, but is not limited to,cytotoxic nucleosides or nucleotides, antisense oligonucleotides,radionuclides, energy absorbing and energy emitting agents, and othercytotoxic agents. Other cytotoxic agents include, but are not limitedto, ricin (or more particularly the ricin A chain), aclacinomycin,diphtheria toxin, Monensin, Verrucarin A, Abrin, Tricothecenes, andPseudomonas exotoxin A, taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, anti-mitotic agentssuch as the vinca alkaloids (e.g., vincristine and vinblastine),colchicin, anthracyclines such as doxorubicin and daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU),lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)(DDP)), and antibiotics, including but not limited to, dactinomycin(formerly actinomycin), bleomycin, mithramycin, calicheamicin, andanthramycin (AMC).

“Cytotoxic nucleoside or nucleotide” as used herein includes, but is notlimited to, 2′,2′-difluorodeoxycytidine, (dFdC, gemcitabine),5-fluorouracil (5-FU), 5-fluoro-2′-deoxyuridine-5′-O-monophosphate(FdUMP), 5-fluoro-2′-deoxyuridine (FdU), arabinosylcytosine (Ara-C),arabinosyl adenosine (Ara-A), fluorouracil arabinoside, mercaptopurineriboside, 5-aza-2′-deoxycytidine, arabinosyl 5-azacytosine,6-azauridine, azaribine, 6-azacytidine,trifluoro-methyl-2′-deoxyuridine, thymidine, thioguanosine,3-deazautidine, 2-Chloro-2′-deoxyadenosine (2-CdA), AZT(azidothymidine), 2′,3′-dideoxyinosine (ddI), cytotoxicnucleoside-corticosteroid phosphodiester, 5-bromodeoxyuridine5′-methylphosphonate, 5-fluorodeoxyuridine (FdUrd), fludarabine(2-F-ara-AMP), 6-mercaptopurine and 6-thioguanine,2-chlorodeoxyadenosine (CdA), 2′-deoxycoformycin (pentostatin),4′-thio-beta-D-arabinofuranosylcytosine, and any other cytotoxic dA, dC,dT, dG, dU, or homologs thereof.

In some embodiments, modified oligonucleotides incorporate activatedanticancer drugs into three-dimensional nucleic acid structures thatselectively bind to and are internalized by cancer cells. Modifiedoligonucleotides are comprised, in part, of relatively low molecularweight activated drugs. Thus, the three-dimensional structures ofmodified oligonucleotides that facilitate selective binding to andpenetration of targeted cells are formed based upon the chemical andstructural properties of the component drugs. In preferred embodiments,the activated drug is 5-fluoro-2′-deoxyuridine-5′-O-monophosphate(FdUMP).

The term “antisense oligonucleotide,” as used herein, refers to anucleic acid that is complementary to and specifically hybridizes to aspecified DNA or RNA sequence. Antisense oligonucleotide includes, butis not limited to, ribozymes, small interfering RNAs, short hairpinRNAs, micro RNAs, triplex-forming oligonucleotides, and/or PNAs.Antisense oligonucleotides and nucleic acids that encode the same can bemade in accordance with conventional techniques. See, e.g., U.S. Pat.No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.Those skilled in the art will appreciate that it is not necessary thatthe antisense oligonucleotide be fully complementary to a targetsequence, as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to specifically hybridize to itstarget and reduce production of the polypeptide (e.g., by at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more).

“Radionuclide” as described herein may be any radionuclide suitable fordelivering a therapeutic dosage of radiation to a tumor or cancer cell,including, but not limited to, ²²⁵Ac, ²²⁷Ac, ²¹¹At, ¹³¹Ba, ⁷⁷Br, ¹⁰⁹Cd,⁵¹Cr, ⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd, ¹⁹⁸Au, ¹⁶⁶Ho, ^(113m)In, ^(115m)In,¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir, ¹⁹²Ir, ¹⁹⁴Ir, ⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu,¹⁰⁹Pd, ³²P, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag,⁸⁹Sr, ³⁵S, ¹⁷⁷Ta, ¹¹⁷ mSn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁶⁹Yb, ⁹⁰Y, ²¹²Bi, ²¹³Bi,¹¹⁹Sb, ¹⁹⁷Hg, ⁹⁷Ru, ¹⁰⁰Pd, ^(101m)Rh, and ²¹²Pb.

“Energy absorbing and energy emitting agent” as used herein includes,but is not limited to, diagnostic agents, contrast agents, iodinatedagents, radiopharmaceuticals, fluorescent compounds and fluorescentcompounds coencapsulated with a quencher, agents containing MRS/MRIsensitive nuclides, genetic material encoding contrast agents, andenergy absorbing and heat emitting nanomaterials including, but notlimited to, single-walled nanotubes and gold nanocages. Some examples ofcontrast agents include, but are not limited to, metal chelates,polychelates, multinuclear cluster complexes (U.S. Pat. No. 5,804,161),halogenated xanthene or a functional derivative of a halogenatedxanthene (U.S. Pat. No. 6,986,740),gadolinium-diethylenetriaminepentaacetic acid (gadopentetatedimeglumine, GdDTPA; Magnavist), gadoteridol (ProHance), gadodiamide,gadoterate meglumine (Gd-DOTA), gadobenate dimeglumine (Gd-BOPTA/Dimeg;MultiHance), mangafodipir trisodium (Mn-DPDP), ferumoxides, paramagneticanalogue of doxorubicin, and ruboxyl (Rb). Some examples of iodinatedagents include, but are not limited to, diatrizoate(3,5-di(acetamido)-2,4,6-triiodobenzoic acid), iodipamide(3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid), acetrizoate[3-acetylamino-2,4,6-triiodobenzoic acid], aminotrizoate[3-amino-2,4,6-triiodobenzoic acid]), and iomeprol. Examples ofradiopharmaceuticals include, but are not limited to, fluorine-18fluorodeoxyglucose ([18F]FDG), Tc-99m Depreotide, carbon-11hydroxyephedrine (HED), [18F]setoperone, [methyl-11C]thymidine, 99mTc-hexamethyl propyleneamine oxime (HMPAO), 99 mTc-L, L-ethylcysteinatedimer (ECD), 99 mTc-sestamibi, thallium 201,I-131metaiodobenzylguanidine (MIBG), 123I—N-isopropyl-p-iodoamphetamine(IMP), 99 mTc-hexakis-2-methoxyisobutylisonitrile (MIBI), 99mTc-tetrofosmin. Examples of agents containing MRS/MRI sensitivenuclides include, but are not limited to, perfluorocarbons andfluorodeoxyglucose. Examples of genetic material encoding contrastagents include, but are not limited to, paramagnetic reporter genes suchas ferredoxin; paramagnetic tag(s) on liposomal lipids such asparamagnetic chelating groups added to PEG; detectable probes; andluciferin/luciferase reporter system.

“Nucleic acid” as used herein refers to single- or double-strandedmolecules which may be deoxyribonucleic acid (DNA), ribonucleic acid(RNA), or homologs thereof such as peptide nucleic acid (PNA), which iscomprised of stretches of nucleic acid polymers linked together bypeptide linkers, or a combination thereof. The nucleic acid mayrepresent a coding strand or its complement. The nucleic acids of thisinvention may be comprised of any combination of naturally-occurringnucleosides (A, G, C, T, U), and/or the nucleic acids may comprisenucleoside or nucleotide analogs and/or derivatives as are well known inthe art, including cytotoxic, synthetic, rare, non-natural bases oraltered nucleotide bases. A nucleic acid molecule in the form of apolymer of DNA may be comprised of one or more segments of cDNA, genomicDNA or synthetic DNA. In addition, a modification can be incorporated toreduce exonucleolytic degradation, such as a reverse (3′→5′) linkage atthe 3′-terminus.

“Cell of interest” as used herein may be any suitable cell, includingbut not limited to cancer cells, tissue cells generally (e.g., muscle,bone, nerve, liver, lung, etc.), pathological and non-pathologicalmicrobial cells (e.g., bacterial, mycobacterial, spirochetalrickettsial, chlamydial, mycoplasmal, and fungal, etc.), parasitic cells(e.g., protozoal, helminth, etc.), and plant cells, etc.

“Cancer cell” as used herein may be any cancer cell, including, but notlimited to, lung, colon, ovarian, prostate, bone, nerve, liver,leukemia, and lymphoma cells.

“Bacterial cell” as used herein may be any bacterial cell including, butnot limited to, Gram-negative bacteria, Gram-positive bacteria and otherbacteria.

Examples of Gram-negative bacteria include, but are not limited to,bacteria of the genera, Salmonella, Escherichia, Klebsiella,Haemophilus, Pseudomonas, Proteus, Neisseria, Vibro, Helicobacter,Brucella, Bordetella, Legionella, Campylobacter, Francisella,Pasteurella, Yersinia, Bartonella, Bacteroides, Streptobacillus,Spirillum, Moraxella and Shigella. Furthermore, bacterial cell ofinterest includes Gram-negative bacteria including, but not limited to,Escherichia coli, Pseudomonas aeruginosa, Neisseria meningitides,Neisseria gonorrhoeae, Salmonella typhimurium, Salmonella entertidis,Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus ducreyi,Proteus mirabilis, Vibro cholera, Helicobacter pylori, Brucella abortis,Brucella melitensis, Brucella suis, Bordetella pertussis, Bordetellaparapertussis, Legionella pneumophila, Campylobacter fetus,Campylobacter jejuni, Francisella tularensis, Pasteurella multocida,Yersinia pestis, Bartonella bacilliformis, Bacteroides fragilis,Bartonella henselae, Streptobacillus moniliformis, Spirillum minus,Moraxella catarrhalis (Branhamella catarrhalis), and Shigelladysenteriae.

Examples of Gram-positive bacteria include, but are not limited to,bacteria of the genera Listeria, Staphylococcus, Streptococcus,Bacillus, Corynebacterium, Peptostreptococcus, and Clostridium.Furthermore, bacterial cell of interest includes Gram-positive bacteriaincluding, but not limited to, Listeria monocytogenes, Staphylococcusaureus, Streptococcus pyogenes, Streptococcus pneumoniae, Bacilluscereus, Bacillus anthracis, Clostridium botulinum, Clostridiumperfringens, Clostridium difficile, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium ulcerans, and Peptostreptococcusanaerobius.

Additional bacteria include bacterial genera including, but not limitedto, Actinomyces, Propionibacterium, Nocardia and Streptomyces.Furthermore, bacterial cell of interest of the present inventionincludes, but is not limited to, Actinomyces israeli, Actinomycesgerencseriae, Actinomyces viscosus, Actinomyces naeslundii,Propionibacterium propionicus, Nocardia asteroides, Nocardiabrasiliensis, Nocardia otitidiscaviarum and Streptomyces somaliensis.

“Mycobacterial cell” as used herein may be any mycobacterial cell,including but not limited to mycobacteria belonging to the mycobacteriafamilies including, but not limited to, Mycobacteriaceae. Additionally,mycobacterial cell of the present invention includes, but is not limitedto, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacteriumavium intracellulare, Mycobacterium kansasii, and Mycobacteriumulcerans.

“Spirochetal cell” as used herein may be any spirochetal cell, includingbut not limited to spirochetes belonging to the genera including, butnot limited to, Treponema, Leptospira, and Borrelia. Additionally,spirochetal cell of the present invention includes, but is not limitedto, Treponema palladium, Treponema pertenue, Treponema carateum,Leptospira interrogans, Borrelia burgdorferi, and Borrelia recurrentis.

“Rickettsial cell” as used herein may be any rickettsial cell, includingbut not limited to rickettsia belonging to the genera including, but notlimited to, Rickettsia, Ehrlichia, Orienta, Bartonella and Coxiella.Furthermore, rickettsial cell includes, but is not limited to,Rickettsia rickettsii, Rickettsia akari, Rickettsia prowazekii,Rickettsia typhi, Rickettsia conorii, Rickettsia sibirica, Rickettsiaaustralis, Rickettsia japonica, Ehrlichia chaffeensis, Orientatsutsugamushi, Bartonella quintana, and Coxiella burni.

“Chlamydial cell” as used herein may be any chlamydial cell belonging tothe genera including, but not limited to, Chlamydia. Furthermore,chlamydial cell of the present invention includes, but is not limitedto, Chlamydia trachomatis, Chlamydia caviae, Chlamydia pneumoniae,Chlamydia muridarum, Chlamydia psittaci, and Chlamydia pecorum.

“Mycoplasmal cell” as used herein may be any mycoplasmal cell belongingto the genera including, but not limited to, Mycoplasma and Ureaplasma.In addition, mycoplasmal cell includes but is not limited to, Mycoplasmapneumoniae, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasmaurealyticum.

“Fungal cell” as used herein may be any fungal cell belonging to thegenera including, but not limited to, Aspergillus, Candida,Cryptococcus, Coccidioides, Tinea, Sporothrix, Blastomyces, Histoplasma,Pneumocystis and Saccharomyces. Additionally, fungal cell of the presentinvention includes, but is not limited to, Aspergillus fumigatus,Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillusnidulans, Candida albicans, Coccidioides immitis, Cryptococcusneoformans, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrixschenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Histoplasmaduboisii, and Saccharomyces cerevisiae.

Compounds of the present invention may also be used to control microbesin industrial fermentation.

“Parasitic cell” as used herein may include any parasitic cell belongingto the genera including, but not limited to, Entamoeba, Dientamoeba,Giardia, Balantidium, Trichomonas, Cryptosporidium, Isospora,Plasmodium, Leishmania, Trypanosoma, Babesia, Naegleria, Acanthamoeba,Balamuthia, Enterobius, Strongyloides, Ascaradia, Trichuris, Necator,Ancylostoma, Uncinaria, Onchocerca, Mesocestoides, Echinococcus, Taenia,Diphylobothrium, Hymenolepsis, Moniezia, Dicytocaulus, Dirofilaria,Wuchereria, Brugia, Toxocara, Rhabditida, Spirurida, Dicrocoelium,Clonorchis, Echinostoma, Fasciola, Fascioloides, Opisthorchis,Paragonimus, and Schistosoma. Additionally, parasitic cell of thepresent invention includes, but is not limited to, Entamoebahistolytica, Dientamoeba fragilis, Giardia lamblia, Balantidium coli,Trichomonas vaginalis, Cryptosporidium parvum, Isospora belli,Plasmodium malariae, Plasmodium ovale, Plasmodium falciparum, Plasmodiumvivax, Leishmania braziliensis, Leishmania donovani, Leishmania tropica,Trypanosoma cruzi, Trypanosoma brucei, Babesia divergens, Babesiamicroti, Naegleria fowleri, Acanthamoeba culbertsoni, Acanthamoebapolyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxisAcanthamoeba hatchetti, Acanthamoeba rhysodes, Balamuthia mandrillaris,Enterobius vermicularis, Strongyloides stercoralis, Strongyloidesfülleborni, Ascaris lumbricoides, Trichuris trichiura, Necatoramericanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Ancylostomabraziliense, Ancylostoma caninum, Uncinaria stenocephala, Onchocercavolvulus, Mesocestoides variabilis, Echinococcus granulosus, Taeniasolium, Diphylobothrium latum, Hymenolepis nana, Hymenolepis diminuta,Moniezia expansa, Moniezia benedeni, Dicytocaulus viviparous,Dicytocaulus filarial, Dicytocaulus arnfieldi, Dirofilaria repens,Dirofilaria immitis, Wuchereria bancrofti, Brugia malayi, Toxocaracanis, Toxocara cati, Dicrocoelium dendriticum, Clonorchis sinensis,Echinostoma, Echinostoma ilocanum, Echinostoma jassyenese, Echinostomamalayanum, Echinostoma caproni, Fasciola hepatica, Fasciola gigantica,Fascioloides magna, Opisthorchis viverrini, Opisthorchis felineus,Opisthorchis sinensis, Paragonimus westermani, Schistosoma japonicum,Schistosoma mansoni, Schistosoma haematobium and Schistosomahaematobium.

“Extracellular surface protein” as used herein may be any extracellularsurface protein including, but not limited to, growth factor receptors,receptor tyrosine kinases, folate hydrolases, GPI-anchored cell surfaceantigens, pumps, and cell surface receptors including, but not limitedto, G-protein coupled receptors, ion channel-linked receptors, andenzyme-linked receptors.

Extracellular surface proteins of interest may be those “differentiallyexpressed” by a targeted cell of interest, in comparison to a cell thatis not to be targeted by a cytotoxic nucleotide. For example, the cancercells differ from normal cells in many respects, including the up- ordown-regulation of numerous genes. Among the genes that aredifferentially regulated in cancer cells are genes that encode proteinsthat are expressed on the extracellular surface. As an example, specificproteins are expressed on the extracellular surface of prostate cancer(PC) cells that are not expressed (or are expressed at very low levels)by normal prostatic epithelial cells and cells from other normaltissues. Extracellular proteins that are expressed exclusively by PCcells are excellent candidates for specific targeting of malignant cellswith anticancer drugs. Cytotoxic oligodeoxynucleotides (ODNs) may beinternalized by malignant cells that express specific ODN receptorproteins (Corrias et al., Biochem. Pharmacol. 55: 1221-1227 (1998)). Theexpression of prostate specific membrane antigen (PSMA) is limited to PCcells and cells of the tumor neovasculature (Schulke et al., Proc. Natl.Acad. Sci. USA 100: 12590-12595 (2003)). A second protein that displayscharacteristics suitable for developing targeted therapeutics for PC isprostate stem cell antigen (PSCA; Saffran et al., Proc. Natl. Acad. Sci.USA 98: 2658-2663 (2001)).

Compounds of the present invention may also be used for the treatment ofa viral disease in a cell of interest. Viral diseases include, but arenot limited to, those caused by viruses belonging to the viral familiesincluding, but not limited to, Flaviviridae, Arenaviradae, Bunyaviridae,Filoviridae, Poxyiridae, Togaviridae, Paramyxoviridae, Herpesviridae,Picornaviridae, Caliciviridae, Reoviridae, Rhabdoviridae, Papovaviridae,Parvoviridae, Adenoviridae, Hepadnaviridae, Coronaviridae, Retroviridae,and Orthomyxoviridae. Furthermore, viral diseases that can be treatedusing the compounds of the present invention can be caused by theviruses including, but not limited to, Yellow fever virus, St. Louisencephalitis virus, Dengue virus, Hepatitis G virus, Hepatitis C virus,Bovine diarrhea virus, West Nile virus, Japanese B encephalitis virus,Murray Valley encephalitis virus, Central European tick-borneencephalitis virus, Far eastern tick-born encephalitis virus, Kyasanurforest virus, Louping ill virus, Powassan virus, Omsk hemorrhagic fevervirus, Kumilinge virus, Absetarov anzalova hypr virus, Ilheus virus,Rocio encephalitis virus, Langat virus, Lymphocytic choriomeningitisvirus, Junin virus, Bolivian hemorrhagic fever virus, Lassa fever virus,California encephalitis virus, Hantaan virus, Nairobi sheep diseasevirus, Bunyamwera virus, Sandfly fever virus, Rift valley fever virus,Crimean-Congo hemorrhagic fever virus, Marburg virus, Ebola virus,Variola virus, Monkeypox virus, Vaccinia virus, Cowpox virus, Orf virus,Pseudocowpox virus, Molluscum contagiosum virus, Yaba monkey tumorvirus, Tanapox virus, Raccoonpox virus, Camelpox virus, Mousepox virus,Tanterapox virus, Volepox virus, Buffalopox virus, Rabbitpox virus,Uasin gishu disease virus, Sealpox virus, Bovine papular stomatitisvirus, Camel contagious eethyma virus, Chamios contagious eethyma virus,Red squirrel parapox virus, Juncopox virus, Pigeonpox virus,Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpoxvirus, Peacockpox virus, Penguinpox virus, Mynahpox virus, Sheeppoxvirus, Goatpox virus, Lumpy skin disease virus, Myxoma virus, Harefibroma virus, Fibroma virus, Squirrel fibroma virus, Malignant rabbitfibroma virus, Swinepox virus, Yaba-like disease virus, Albatrosspoxvirus, Cotia virus, Embu virus, Marmosetpox virus, Marsupialpox virus,Mule deer poxvirus virus, Volepox virus, Skunkpox virus, Rubella virus,Eastern equine encephalitis virus, Western equine encephalitis virus,Venezuelan equine encephalitis virus, Sindbis virus, Semliki forestvirus, Chikungunya virus, O'nyong-nyong virus, Ross river virus,Parainfluenza virus, Mumps virus, Measles virus (rubeola virus),Respiratory syncytial virus, Herpes simplex virus type 1, Herpes simplexvirus type 2, Varicella-zoster virus, Epstein-Barr virus,Cytomegalovirus, Human b-lymphotrophic virus, Human herpesvirus 7, Humanherpesvirus 8, Poliovirus, Coxsackie A virus, Coxsackie B virus,ECHOvirus, Rhinovirus, Hepatitis A virus, Mengovirus, ME virus,Encephalomyocarditis (EMC) virus, MM virus, Columbia SK virus, Norwalkagent, Hepatitis E virus, Colorado tick fever virus, Rotavirus,Vesicular stomatitis virus, Rabies virus, Papilloma virus, BK virus, JCvirus, B19 virus, Adeno-associated virus, Adenovirus, serotypes 3, 7,14, 21, Adenovirus, serotypes 11, 21, Adenovirus, Hepatitis B virus,Coronavirus, Human T-cell lymphotrophic virus, Human immunodeficiencyvirus, Human foamy virus, Influenza viruses, types A, B, C, andThogotovirus.

Additionally, compounds of the present invention may be used as anherbicide. The compounds of the present invention may be applied to thesurface of the plant including, but not limited to, leaves, stems,flowers, fruits, roots, cells or callus tissue. Alternatively, thecompounds of the present invention may be introduced into the plant viamethods standard in the art including, but not limited to,microinjection, electroporation, particle bombardment, andAgrobacterium-mediated transformation.

Further, compounds of the present invention may also be used fortreatment of infection of plants and plant cells by plant pathogens, theplant pathogens including, but not limited to, bacteria, fungi,oomycetes, viruses, and nematodes. For the purpose of treatment of plantpathogenic infections, the compounds of the present invention may beapplied to the surface of a plant including, but not limited to, leaves,stems, flowers, fruits, roots, cells or callus tissue. Alternatively,the compounds of the present invention may be introduced into the plantvia methods standard in the art including, but not limited to,microinjection, electroporation, particle, bombardment, andAgrobacterium-mediated transformation.

As used herein, the term “treat” or “treatment” refers to an actionresulting in a reduction in the severity of the subject's condition,wherein the condition is at least partially improved or ameliorated,and/or there is some alleviation, mitigation or decrease in at least oneclinical symptom (or agricultural index for plants), and/or there is adelay in the progression of the condition, and/or prevention or delay ofthe onset of the condition. Thus, the term “treat” refers to bothprophylactic and therapeutic treatment regimes. Compounds generated bythe methods of the present invention may be used for the diagnosisand/or treatment of human subjects, or animal subjects for veterinary ordrug development purposes. Examples of animal subjects include mammalian(e.g., dog, cat, mouse, rat, horse, cow, pig, sheep, etc.), reptile,amphibian, and avian (e.g., parrot, budgie, chicken, turkey, duck,geese, quail, pheasant) subjects.

A first pool of nucleic acids used to carry out the present inventionmay be comprised of range of about 10⁶, 10⁸, 10¹⁰, 10¹¹, 10¹², or 10¹³,to a range of about 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁸, 10²⁰, 10²¹, 10²², or 10²³nucleic acid species. In some embodiments, the first pool of nucleicacids comprises about 10¹⁰ to 10¹⁸ nucleic acid species. In furtherembodiments, the first pool of nucleic acids comprises about 10¹³ to10¹⁴ nucleic acid species. In still further embodiments, the first poolof nucleic acids comprises 10¹⁵ nucleic acid species.

The size of the nucleic acids species within the first pool can be in arange of about 30 nucleotides to about 150 nucleotides. In preferredembodiments, the nucleic acid species of the present invention comprisesthree regions: a “random” region flanked by two “constant” regions, asillustrated in FIG. 2. The two “constant” regions, A and B, need not beidentical to each other, but comprise known nucleotide sequences. These“constant” regions are used for the annealing of PCR primers during PCRamplification. The lengths of the “constant” regions of the presentinvention can be in a range of about 8 nucleotides to about 35nucleotides. In some embodiments the lengths of the “constant” regionsare in a range of about 12 nucleotides to about 22 nucleotides. Thelength of Region A need not be the same as the length of Region B, andindeed each region may be modified in length and/or sequence based onfolding predictions or results following the identification of optimal“random” regions.

The “random” region of the nucleic acids species within the first poolconsists of random arrangements of nucleotide sequences. Those “random”regions that selectively bind to a target of interest are selected forduring the selection of a first subpopulation of interest, and becomethe second pool of nucleic acid species. However, in some embodimentsthere is some predetermined bias contained within the “random” regions.The predetermined bias may be performed to facilitate the inclusion ofparticular cytotoxic nucleotides. For instance, the first pool ofnucleic acids may include a greater representation of a particularnucleoside (A, C, G, T, U). In another embodiment, the nucleic acid poolmay include a lesser representation of a particular nucleoside (A, C, G,T, U). In a further embodiment, the nucleic acid pool may include aspecific sequence element that may confer antisense or antigeneproperties to all of the members of the resulting subpopulation.

In some embodiments, the lengths of the nucleic acids species within thefirst pool includes the “constant” regions, wherein the size of thenucleic acids species can be in a range of about 45 nucleotides to about130 nucleotides in length. In other embodiments, the size of the nucleicacids species within the first pool can be in a range from about 60nucleotides to about 100 nucleotides in length. In further embodiments,the size of the nucleic acids species within the first pool can be in arange from about 65 nucleotides to about 80 nucleotides in length. Anadditional embodiment of the present invention comprises a first pool ofnucleic acid species, wherein the size of the nucleic acid species isabout 70 nucleotides in length.

In some embodiments, the lengths of the nucleic acids species within thefirst pool does not include the “constant” regions, wherein the size ofthe nucleic acids species can be in a range of about 20 nucleotides toabout 105 nucleotides in length. In other embodiments of the presentinvention, the size of the nucleic acids species within the first poolcan be in a range from about 35 nucleotides to about 75 nucleotides inlength. In further embodiments, the size of the nucleic acids specieswithin the first pool can be in a range from about 40 nucleotides toabout 55 nucleotides in length. An additional embodiment of the presentinvention comprises a first pool of nucleic acid species, wherein thesize of the nucleic acids species is about 45 nucleotides in length. Astill further embodiment of the present invention comprises a first poolof nucleic acid species, wherein the size of the nucleic acid species isabout 30 nucleotides in length.

In some embodiments of the present invention, the first pool of nucleicacid species comprises about 10¹³ to 10¹⁴ nucleic acid species, whereinthe nucleic acid species are about 70 nucleotides in length includingthe “constant” regions. In further embodiments, the first pool ofnucleic acid species comprises about 10¹⁵ nucleic acid species, whereinthe nucleic acid species are about 70 nucleotides in length includingthe “constant” regions.

In some embodiments of the present invention, the first pool of nucleicacid species comprises about 10¹³ to 10¹⁴ nucleic acid species, whereinthe nucleic acid species are about 30 nucleotides in length notincluding the “constant” regions. In a further embodiment, the firstpool of nucleic acid species comprises about 10¹⁵ nucleic acid species,wherein the nucleic acid species are about 30 nucleotides in length notincluding the “constant” regions.

The step of combining the first pool of nucleic acids with theextracellular surface protein may be random or it may be performed withsome predetermined bias. The predetermined bias may be performed tofacilitate the inclusion of particular cytotoxic nucleotides. In oneembodiment, the first pool of nucleic acids may include a greaterrepresentation of a particular nucleoside (A, C, G, T, U). In anotherembodiment, the nucleic acid pool may include a lesser representation ofa particular nucleoside (A, C, G, T, U). In a further embodiment, thenucleic acid pool may include a specific sequence element that mayconfer antisense or antigene properties to all of the members of theresulting subpopulation. This predetermined bias may be found in anyportion of the nucleic acids, including the constant region A, randomregion B, and/or constant region C (See FIG. 2).

The step of selecting a first subpopulation of nucleic acids from thefirst pool, wherein the first subpopulation comprises at least onenucleic acid that binds specifically to the extracellular surfaceprotein of interest, may be done using any method standard in the art,including, but not limited to, such methods as affinity chromatography,capillary eletrophoresis, field flow fractionation chromatography andsurface plasmon resonance. The methods of capillary electrophoresis andfield flow fractionation chromatography may be further combined withmass spectrometry to obtain sequence information on the selected firstsubpopulation of nucleic acids. The step of selecting may be performedonce, or the nucleic acids from the first pool may be subjected toadditional rounds of selection to identify those nucleic acids with highaffinity for the extracellular surface protein of interest.

“Amplification” or “amplify” as used herein means the construction ofmultiple copies of a nucleic acid sequence, or multiple copiescomplementary to the nucleic acid sequence, using at least one of thenucleic acid sequences as a template. The step of amplifying nucleicacids may be any method standard in the art for amplifying nucleic acidsincluding, but not limited to, polymerase chain reaction (PCR),self-sustained sequence replication, strand-displacement amplification,“branched chain” DNA amplification, ligase chain reaction (LCR) andQ-Beta replicase amplification (QBR). In some embodiments of the presentinvention, the selected nucleic acids are amplified using PCR.

The step of selecting a second subpopulation comprising at least onenucleic acid species from the first subpopulation, wherein the at leastone nucleic acid species is internalized by said cell of interestincludes, but is not limited to, such detection methods as fluorescencemicroscopy and flow cytometry, including, but not limited to,fluorescent-activated cell sorting.

To aid in detection, the at least one nucleic acid from the firstsubpopulation may be labeled with a detectable label using methodsstandard in the art, wherein the dectable label can include, but is notlimited to, fluorescent dyes, fluorophores, chromophores, affinitylabels, metal chelates, chemically reactive groups, enzymes,radionuclides, electrochemically detectable moieties, and energyabsorbing or energy emitting compounds.

Fluorescent dyes that can be used with the present invention are anycapable of binding to nucleic acids as defined herein and include, butare not limited to, the coumarin dyes, acetyl azide, fluoresceinisothiocyanate, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,8-(6-aminohexyl)aminoadenosine 3′,5′-cyclicmonophosphate,bis(triethylammonium) salt, rhodamine dyes, sulfonyl chloride, CyDye™fluors, and carboxynaphtofluorescein. The haptenes that may be used forlabeling include, but are not limited to, biotin, digoxigenin, and2,4-dinitrophenyl. The haptenes require fluorescently-labeled antibodiesor specific proteins for visualization/detection.

Labeling of nucleic acids with electrophore mass labels is described,for example, in Xu et al., J. Chromatography 764:95-102 (1997).Electrophores are compounds that can be detected with high sensitivityby electron capture mass spectrometry (EC-MS). Electrophore mass labelscan be attached to a probe using chemistry that is well known in the artfor reversibly modifying a nucleotide (e.g., well-known nucleotidesynthesis chemistry teaches a variety of methods for attaching moleculesto nucleotides as protecting groups). Electrophore mass labels aredetected using a variety of well-known electron capture massspectrometry devices. Further, techniques that may be used in thedetection of electrophore mass labels include, for example, fast atomicbombardment mass spectrometry (See Koster et al., Biomedical Environ.Mass Spec. 14:111-116 (1987)); plasma desorption mass spectrometry;electrospray/ionspray (See Fenn et al., J. Phys. Chem. 88:4451-59(1984), PCT Appln. No. WO 90/14148, Smith et al., Anal. Chem. 62:882-89(1990)); and matrix-assisted laser desorption/ionization (Hillenkamp etal. Biological Mass Spectrometry (Burlingame and McCloskey, eds.),Elsevier Science Pub., Amsterdam, pp. 49-60, 1990); Huth-Fehre et al.,Rapid Communications in Mass Spectrometry, 6:209-13 (1992)). (See alsoU.S. Pat. No. 6,979,548 issued to Ford et al.)

Methods for conjugation of detectable labels to nucleic acids are wellknown in the art, for example, Schubert et al., Nucleic Acids Research18:3427 (1990) Smith et al., Nature, 321:674-679 (1986); Agarawal etal., Nucleic Acids Research, 14:6227-6245 (1986); Chu et al., NucleicAcids Research, 16:3671-3691 (1988).

Sequencing at least one selected nucleic acid from a secondsubpopulation may be done according to methods standard in the artincluding, but not limited to, automated nucleic acid sequencingprocedures as disclosed in Naeve, C. W., (1995) Biotechniques 19:448,and sequencing by mass spectrometry. See, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162(1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159(1993).

Incorporation of a compound of interest into a selected nucleic acidsequence requires that the selected nucleic acid sequence retains itsoriginal three-dimensional structure of the native sequence followingthe incorporation. In some embodiments, folding calculations areperformed to compare the predicted folding patterns of the chemicalstructure of the native nucleic acid sequence with that of a nucleicacid sequence incorporating one or more compound of interest.Calculations can be performed with, e.g., folding programs such as mFOLD(Michael Zuker, Burnet Institute). Such calculations apply an algorithmto the native sequence of the nucleic acid to determine folding patternsthat yield the most stable secondary structures. This approach providesinsight into the likely location of double helical regions that occurwithin the three-dimensional structure of the nucleic acid. Thestructural characteristics of the native and modified nucleic acids canalso be determined using circular dichroism (CD) spectroscopy andultraviolet (UV) hyperchromicity measurements. Other methods ofcomparison will be apparent to those skilled in the art. Preferrednucleic acids of interest are those that incorporate compounds ofinterest in such a way as to not significantly alter the foldingcharacteristics of the native sequences.

In some embodiments, modified nucleic acids are further evaluated forthe extent to which they selectively kill cells of interest, e.g.,through the release of cytotoxic nucleotides by 3′-O-exonucleolyticdegradation. Cell viability can be evaluated, e.g., using3-(4,5-dimethylhiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assays. Preferred nucleic acids are those that arecytotoxic towards cells of interest and not cytotoxic to non-targetedcells.

Synthesizing a nucleic acid having a sequence corresponding to aselected nucleic acid and incorporating a compound of interest may doneaccording to any method standard in the art including, but not limitedto, de novo chemical synthesis of polynucleotides, such as by presentlyavailable automated DNA synthesizers, and standard phosphoramiditechemistry. De novo chemical synthesis of a polynucleotide can beconducted using any suitable method, including, but not limited to, thephosphotriester or phosphodiester methods. See Narang et al., Meth.Enzymol., 68:90 (1979); U.S. Pat. No. 4,356,270; Itakura et al., Ann.Rev. Biochem., 53:323-56 (1989); Brown et al., Meth. Enzymol., 68:109(1979); and U.S. Pat. No. 6,911,310 issued to Heller. In one embodimentof the present invention, automated nucleic acid synthesis is conductedusing an Applied Biosystem 394™ automated DNA/RNA synthesizer (AppliedBiosystems, Foster City, Calif.).

In some embodiments of the present invention, a synthetic nucleic acidmay comprise one compound of interest. In other embodiments, a syntheticnucleic acid may incorporate more than one compound of interest. In someembodiments, one of the compounds of interest incorporated into thesynthetic nucleic acid may be a detectable compound, and/or an activecompound.

In preferred embodiments, cytotoxic oligodeoxynucleotides areoligodeoxynucleotides (ODNs) that contain one or more cytotoxicnucleoside analogs. Once incorporated into an ODN, the5′-O-monophosphate form of the nucleoside is present as an intact unitthat is embedded in the ODN polymer. The cytotoxic nucleoside analogsmay be incorporated as a stretch of several ODNs, or may be incorporatedat various places in the nucleotide species. In some embodiments,cytotoxic ODNs are arranged as a stretch of 2, 3, 4, or 5 to 20, 25, 30,or 40 ODNs. A preferred example of a cytotoxic ODN is FdUMP[10], alinear homopolymer of FdUMP, the thymidylate synthase inhibitorymetabolite of the anticancer drug 5-fluorouracil (5FU). (Gmeiner, Curr.Med. Chem. 12: 1345-1359 (2005); Gmeiner et al., Nucl. Nuct. Nucl. Acids23: 401-410 (2004)). Another preferred example is FdUMP[5], illustratedin FIG. 1. The cytotoxic ODNs may be included in the synthesis of adesired nucleotide species, or may be appended to a desired nucleotidespecies. Synthesis and toxicity of FdUMP are found in U.S. Pat. Nos.5,457,187 (Gmeiner et al.); 5,614,505 (Gmeiner et al.); 5,663,321(Gmeiner et al.); 5,741,900 (Gmeiner et al.); and 6,342,485 (Gmeiner).

In some embodiments, ODNs may be synthesized to incorporate compounds ofinterest such as cytotoxic nucleoside analogs, either before or afterthe enrichment selections of candidate ODN sequences. In a preferredembodiment, ODNs selected in the first and second pools do not comprisea compound of interest. The selected ODNs are sequenced and analyzed todetermine whether the incorporation of a compound on interest willaffect their activity towards a biological target of interest. CytotoxicODNs are then subsequently synthesized consistent with analysispredictions (e.g. predicted folding). However, synthesis of ODNscontaining a compound on interest such as a cytotoxic nucleoside analogmay also be performed prior to the enrichment steps.

Modified ODNs of the present invention can be optimized, e.g., fortreatment of PC and other malignancies. In some embodiments of thepresent invention, the modified ODNs target xPSM using FdUMP as theactive drug. In other embodiments, the modified ODNs targetextracellular surface proteins that are differentially expressedspecifically on the surface of certain PC cells (e.g. prostate stem cellantigen). In further embodiments the modified ODNs administered to aparticular patient may be customized to reflect the protein profileexpressed by a specific patient. Additionally, the choice of drugs forinclusion into the modified ODN structure may be expanded to reflect thedrug-profile that provides the maximum response for a particularmalignancy. The ODNs of the present invention are compatible with awide-range of cytotoxic compounds, including, but not limited to,nucleoside analogs, cytotoxic drugs, radionuclides, modifiers of geneexpression and nanoparticles.

EXAMPLE 1 Identification of Oligodeoxynucleotide Sequences that UndergoFacilitated Uptake into Prostate Cancer Cells as a Consequence ofBinding to the Extracellular Domain of Prostate Specific MembraneAntigen

In order to identify oligodeoxynucleotide (ODN) sequences that bind tothe extracellular domain of prostate specific membrane antigen (xPSM)and undergo facilitated uptake into PC cells as a consequence of xPSMbinding, a pool of random ODN sequences that is sufficiently large andcomplex was used. Within this pool, the ODN sequences fold into athree-dimensional structure. Certain species within this pool bind xPSMwith high affinity. Rounds of selective binding of ODNs to anxPSM-affinity matrix are used to identify sequences of interest. ODNsequences that bind xPSM with high affinity are amplified using PCR. ODNsequences that undergo selective facilitated uptake into PC cells, suchas those of LNCaP cell line, are candidate cytotoxic ODNs (i.e., the ODNsequences that contain cytotoxic nucleotides and that selectively enterand kill PC cells).

A. Preparation of xPSM Affinity Matrix. A protocol was designed toexpress the 706 amino acids comprising the extracellular domain of PSMAfrom Sf-9 (insect) cells using baculovirus. Pelleted LNCaP cells (quickfrozen in liquid N₂) were used as the source of RNA for cloning of PSMA.RNA was extracted from the pelleted cells and the integrity of the RNAwas verified by gel electrophoresis. First strand cDNA synthesis wasaccomplished by reverse transcription using oligo dT(N) and randomhexamer primers, and SENSISCRIPT™ reverse transcriptase (Qiagen). ThecDNA was purified using a Qiagen column. Primers were designed bystandard procedures for cloning of the extracellular domain of PSMA intothe pBacGus3 plasmid (Novagen). The 5′-primer includes an XbaI site tofacilitate cloning into the NheI site in the vector (pBacGus3). The3′-primer includes a HindIII site for cloning into the HindIII site ofpBacGus3. The 3′-primer was also designed to express a His-tag sequencein the vector to attach the protein to magnetic beads for use as anaffinity matrix. The target region of the PSMA gene was amplified fromcDNA by PCR using PHUSION™ high-fidelity DNA polymerase (New EnglandBiolabs) to yield a product of the expected length (˜2.2 kB). The PCRproduct was successfully cloned into the pBacGus3 shuttle vector with 6out of 6 colonies showing the insert.

Procedures for the expression of human xPSM from Sf-9 cells usingbaculovirus are known in the art. The His-tagged xPSM protein was boundto M450 magnetic beads (Dynal Biotech). The suitability of the xPSMbeads for ODN selection was verified by determining that a known RNAaptamer sequence to xPSM binds to the beads (Lupold et al., Cancer Res.62: 4029-4033 (2002)). The xPSM-beads were then used for theidentification of ODNs that bind xPSM with high affinity, using theprocedures described in the following section.

B. Selection of ODNs that Bind xPSM with High Affinity. The exponentialenrichment methodology used to identify ODNs with high binding affinityfor xPSM has been described previously (Morris et al., Proc. Natl. Acad.Sci. USA 95: 2902-2907 (1998); FIG. 2). A single-stranded DNA librarywas prepared on a 2 micromol scale containing a 45 nucleotide (45mer)random sequence flanked by two 21 nucleotide constant regions. One ofthe two constant regions that flank the random 45mer region of the DNAlibrary construct consists of predominantly A-nucleotides and results inthe ODN sequences selected for xPSM binding affinity having dT-residuespredominantly in the 3′-terminus. Thus, constant region A is a 21merfixed sequence, random region B is a 45mer variable sequence, andconstant region C is a 21mer fixed sequence that is adenine-rich (seeFIG. 2). T-rich primers used for PCR amplification that arecomplementary to the A-rich constant region are biotinylated at the 5′terminus. The amplified DNA is purified on streptavidin beads to obtainsingle-stranded ODNs.

The single-stranded DNA library was gel-purified to yield 6.9 nmol ofmaterial (4.16×10¹⁵ molecules). The single-stranded material was nextconverted to dsDNA using a series of “fill-in” reactions with T7 DNApolymerase. These “fill-in” reactions were each run using 2 μg of thessDNA library, a 1.5-fold excess of the 21 nucleotide primercomplementary to the 3′-region of the ssDNA, 1.5 μL of 10 mM dNTPs, 5units of T7 DNA polymerase (Fermentas), and 5 μL of 10× reaction buffer(400 mM Tris-HCl, 100 mM MgCl₂, 10 mM DTT), in a 50 μL total reactionvolume. Reactions were incubated for 2 minutes at 37° C. and stopped byheating the reaction to 70° C. for 10 minutes. dsDNA was recovered byethanol precipitation, analyzed by agarose gel electrophoresis, andquantified by UV absorption at 260 nm. Gel electrophoresis confirmedthat essentially all of the ssDNA was converted to dsDNA using the T7DNA polymerase “fill-in” reaction.

The dsDNA (0.35 nmol (2×10¹⁴)) was amplified for 5 cycles by PCR. EachPCR reaction includes 42.6 ng of template DNA and ˜50-fold excess of theprimers complementary to the 3′-terminus of each strand. The primer forthe DNA strand produced in the T7 DNA polymerase “fill-in” reaction isbiotinylated so that amplification of the original ssDNA containing the30mer random sequence will yield biotinylated DNA that can be separatedusing Streptavidin agarose.

For forward rounds of selection, 5-10 μg of annealed ssDNA in 100 μL of100 mM NaCl, 20 mM Tris, 2 mM MgCl₂, 5 mM KCl, 1 mM CaCl₂ with 0.02%Tween-20 were incubated together with the affinity matrix for 60 min at37° C. The supernatant was then removed. Bound material is eluted byheating to 95° C. in the presence of 20 μL of 5 μM 5′-phosphorylatedprimer followed by removal of the supernatant. The ssDNA that boundtightly to the magnetic beads was then converted to dsDNA using the T7fill-in procedure and then ethanol precipitated. The material is thenamplified by PCR and converted to ssDNA using exonuclease λ. Forcounter-selection, 5-10 μg of annealed ssDNA were incubated withmagnetic beads without PSMA.

The sequences of ssDNA that resulted from multiple rounds of selectionare determined by converting the material to dsDNA using a T7 fill-inreaction and then cloning the dsDNA into the pGEM T-Easy vector(Promega). dsDNA for the cloning procedure was gel-purified on a 2%agarose gel and purified using the SV wizard gel clean-up kit (Promega).The pGEM plasmid was then transformed into competent BL21 E. coli cellsand screened for β-galactosidase activity. Sequences are determinedaccording to standard methods of sequencing nucleic acids.

C. Selective Uptake of ODNs Into PC Cells. ODNs selected for highbinding affinity to xPSM are further evaluated for selective binding toPC cells that express xPSM. Fluorescence microscopy is used toinvestigate the selective binding of ODNs to PC cells that express xPSM,such as LNCaP, relative to those that do not express xPSM (e.g., PC-3cells). The ODN sequences are chemically synthesized and 5′-conjugatedwith a fluorescent dye, such as rhodamine, using methods similar tothose previously described (Lupold et al., Cancer Res. 62: 4029-4033(2002)). The binding and uptake of fluorescent-conjugated ODNs into PCcells is determined using a Zeiss confocal microscope. In addition,cellular uptake is quantified using ³²P-labeled ODNs. The time-dependentaccumulation of ³²P-labeled ODN in PC cellular lysates is detected usinga scintillation counter.

EXAMPLE 2 Analysis, Evaluation and Testing of the Selective Cytotoxicityof Modified ODNs Towards PC Cells

A. Analysis of Modified ODN Sequences. ODN sequences that are identifiedbased upon selective facilitated uptake into LNCaP cells are synthesizedwith cytotoxic nucleotides (5-fluoro-2′-deoxyuridine (FdU)) in place ofnative nucleotides (Gmeiner et al., Nucl. Nuct. Nucl. Acids 23: 401-410(2004)). Because introducing non-native nucleotides into the ODNsequence may alter the affinity for xPSM, the affinity of thesubstituted ODN relative to the native sequence is evaluated usinggel-shift assays (Ferber et al., Anal. Biochem. 244: 312-320 (1997)). Ifintroduction of FdU at certain sites substantially reduces affinity ofthe ODN for xPSM, NMR and/or X-ray studies are performed to identifysites for substitution that are less likely to affect binding affinity.

B. Synthesis of Candidate Modified ODNs. The chemical synthesis ofmodified aptamers is performed using an automated DNA synthesizer at acommercial DNA synthesis facility. The FdU-phosphoramidite issynthesized using methods previously described (Gmeiner et al., Nucl.Nuct. Nucl. Acids 23: 401-410 (2004)).

The RNA aptamer A10-3 was synthesized. A10-3 is a 56mer RNA sequencethat binds the extracellular domain of PSMA (xPSM) with high affinity.The sequence for A10-3 (herein “parent aptamer”) is5′rGGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCAC UCCUUGUCAAUCCUCAUCGGC-3′ (SEQ IDNO:1). The 3′-terminus of A10-3 was modified with FdUMP to engineercytotoxicity into the aptamer. Specifically, a modified aptamercontaining the parent A10-3 sequence with 9 FdU nucleotides and one dCnucleotide appended to the 3′-terminus (A10-3: FdU[9]dC) was prepared.The dC nucleotide was required for synthesis of this RNA-DNA hybridmodified aptamer. The negative control was a modified aptamer with dT inplace of FdUMP (A10-3: dT[10]).

For studies evaluating the specific uptake of the modified aptamers intotargeted (LNCaP) versus non-targeted PC cells (PC3), modified aptamerswith fluorescent dyes (either FAM or HEX) at the 5′-terminus wereprepared. These modified aptamers included a reverse (3′→5′) linkage atthe 3′-terminus to reduce exonucleolytic degradation (5′-FAM-A10-3 and5′-FAM-A10-3: dT[10]).

Modified aptamers that have been prepared include:

(a) A10-3:FdU[9]dC. This modified aptamer with 9 FdU nucleotides and onedC nucleotide appended to the 3′-terminus was synthesized to evaluateselective cytotoxicity towards targeted cancer cells.

(b) A10-3-T₄. This modified aptamer with four T deoxynucleotidesreplacing four U ribonucleotides in the aptamer sequence was synthesizedto determine if deoxynucleotide substitution would change the threedimensional structure of the aptamer.

(c) A10-3-FdU₄. This modified aptamer with four FdU deoxyribonucleotidesreplacing four U ribonucleotides in the aptamer sequence was synthesizedto determine if FdU substitution at internal positions of the aptamersequence would result in selective cytotoxicity towards targeted cancercells.

(d) A10-3-FdU₄:FdU[9]dC. This modified aptamer includes a total of 13FdU deoxyribonucleotides (four at internal positions and nine appendedat the 3′-terminus). This is used to evaluate selective cytotoxicitytowards targeted cancer cells.

(e) A “scrambled” version of the A10-3 RNA aptamer sequence (SCR). Thissequence has the same nucleotide composition and length as A10-3, buthas the nucleotides in a different order. The SCR sequence is: (SEQ IDNO: 2) 5′ rCAGGCAUGCCUAGCUAAGCAGCCCAUGGCUUAUGCGC GGAAUAUUGGCUUCCGUUC

(f) 5′-FAM-SCR:T[10]. This modification of the scrambled aptamersequence included a fluorescent dye and 10 T deoxynucleotides appendedat the 3′-terminus. The scrambled aptamer sequence was used as anegative control to verify that the native aptamer structure wasrequired for selective uptake into targeted cancer cells.

(f) SCR:FdU[9]dC. This modified scrambled aptamer sequence was used todetermine the extent that the native aptamer structure was required forselective cytotoxicity towards targeted cancer cells.

C. Folding Analysis of Candidate Modified Aptamers. Engineeringcytotoxicity into an existing aptamer sequence, such as A10-3, requiresthat the aptamer retain the original three-dimensional structurefollowing the substitution of cytotoxic drugs into the native sequence.To gain insight into the structure of A10-3, mFOLD calculations wereperformed. The mFOLD calculations apply an algorithm to the primarysequence of the RNA to determine folding patterns that yield the moststable secondary structures. This approach provides insight into thelikely location of double helical regions that occur within thethree-dimensional structure of the A10-3 aptamer.

The two lowest energy folds identified by the mFOLD program for theA10-3 aptamer are shown in FIGS. 3 (A and B). Both of the lowest energysecondary structures contain a helical region consisting of nine basepairs with a single bulged A. Both of the lowest energy structures alsocontain a short stem loop consisting of four base pairs and a five-baseloop. The major difference between the two lowest energy structures isfor the 16 nucleotides at the 3′-terminus of the sequence. The lowestenergy structure predicts no base pairing between nucleotides in thisregion, while the second lowest energy structure predicts base pairingbetween nucleotides 41-43 and the three nucleotides at the 5′-terminus.Based on this analysis of secondary structure motifs for the A10-3sequence, appending deoxynucleotides to the 3′-terminus of A10-3 waspredicted to have an insignificant effect on the double helicesimportant for the three-dimensional structure formation of A10-3. Italso appeared likely that substitution of U ribonucleotides near the3′-terminus with FdU deoxynucleotides would have minimal effect on theA10-3 structure (FIG. 3C). Subsequent circular dichroism (CD) studies ofT- and FdU-substituted aptamers revealed that insertion of thesedeoxyribonucleotides had minimal effect on aptamer structure orstability.

D. Circular Dichroism and Ultraviolet Analysis of Candidate ModifiedAptamers. The structural characteristics of the parent aptamer andmodified aptamers were determined using circular dichroism (CD)spectroscopy and ultraviolet (UV) hyperchromicity measurements. The datafor the CD spectroscopy are shown in FIGS. 4A (modified aptamer) and 4B(parent aptamer). The circular dichroism curve acquired before thermalmelting is in blue and that obtained post-melt is green. A maximumpositive ellipticity is observed at 270 nm consistent with a highlyfolded structure, probably consisting of A-form helices. The circulardichroism spectra for the modified aptamers are indistinguishable fromcircular dichroism spectra for the parent aptamer. The UV thermal meltdata is shown in FIGS. 4C (modified aptamer) and 4D (parent aptamer).The melting temperature for parent aptamer and the modified aptamers isabout 55° C. Both the parent aptamer and the modified aptamers adopthighly folded structures that are stable at 37° C. These data indicatethat the modification of the aptamer sequence had minimal effect onstructure and thus was not likely to effect binding to the PSMA targetor cellular uptake in cells that express PSMA.

E. Internalization of Modified Aptamers By Targeted Cancer Cells. Thespecific uptake of modified aptamers into targeted cancer cells wasobserved using fluorescence microscopy. The human prostate cancer cellline LNCaP was used as the cellular target for the modified aptamersbecause it is known to express xPSM. PC3 (human prostate cancer) cellswere used as the negative control as they are known not to express xPSM.Cells were grown to the desired level of confluency (˜50%) in 35 mmdishes. The fluorescently labeled modified aptamer (with and without thedT[10] tail) was then added to the medium at 1 micromolar concentration,and the cells were incubated for various time intervals (e.g. 15 min, 30min, 45 min, etc.). The medium containing the dye was then removed, andthe cells were washed with culture medium. Following the washes, thecells were placed in PBS and viewed under a Zeiss Axiovert confocalmicroscope in the core microscopy laboratory of the Comprehensive CancerCenter at Wake Forest University. The results are shown in FIG. 5.

FIG. 5 shows fluorescence images of LNCaP cells following 15 min (B) or45 min (D) exposure to the modified aptamer. The fluorescent aptamer waslocalized predominantly in the nucleus. The corresponding phase contrastimages are shown in FIGS. 5A and 5C, respectively. FIGS. 5E and 5F,respectively, show the phase contrast and fluorescence microscopy imagesof PC3 cells following 30 min exposure to the modified aptamer. Littleuptake of the aptamer is observed in healthy PC3 cells. All cells wereexposed to approximately 1 micromolar concentration of the fluorescentmodified aptamer for the indicated times. Uptake of 5′-FAM-A10-3:T[10]occurred rapidly into nearly all LNCaP cells. Substantial uptake wasobserved with as little as 15 minutes of exposure into these cells.5′-FAM-A10-3:T[10] localized mainly in the nuclei of LNCaP cells. Incontrast, uptake of 5′-FAM-A10-3:T[10] was limited to only a smallpercentage of PC3 cells and these cells appeared to be among the leastrobust upon inspection by phase contrast microscopy. No specificsub-cellular localization of 5′-FAM-A10-3:T[10] was observed in PC3cells. These results demonstrate that modified aptamers, such as5′-FAM-A10-3:T[10], are rapidly and selectively taken up by PC cellsthat express PSMA on the extracellular surface of the plasma membrane.

F. Cytotoxicity of Candidate Modified Aptamers. The candidate modifiedODNs are evaluated for the extent to which they selectively kill PCcells through the release of cytotoxic nucleotides by3′-O-exonucleolytic degradation. The ODN sequences are synthesized usingstandard automated methods with phosphoramidites derived from cytotoxicnucleosides used in place of the native nucleotides. For example, at anylocation in a particular ODN sequence where a dT is located, FdU isinserted. Cytotoxic analogs of dA, dC, and dG may also be inserted inplace of these native nucleotides. The cytotoxicity of these candidatemodified ODNs is evaluated with regard to PC cells that express xPSM(e.g. LNCaP), to PC cells that do not express xPSM (e.g. PC3), and tonormal prostatic epithelial cells. Uptake kinetics for modified ODNs arecalculated from the time- and concentration-dependence of ³²P-labeledmodified aptamers in target cells. The mechanisms for the cytotoxicityof the modified ODNs that are investigated include thymidylate synthaseinhibition, nucleotide pool imbalance, and DNA damage using methodspreviously described (Gmeiner et al., Nucl. Nuct. Nucl. Acids 23:401-410 (2004)).

The cytotoxicity of candidate modified aptamers towards LNCaP and PC3cells is evaluated using3-(4,5-dimethylhiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assays. Cells are grown in RPMI 1640 medium (LifeTechnologies, Rockville, Md.) supplemented with 10% FBS, 5 mM glutamate,penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37° C. in a 5% CO₂atmosphere. The cytotoxicity of the candidate modified aptamers isevaluated over the concentration range 10⁻¹²-10⁻⁶ M with 48 h drugexposure. Cell viability is assessed using the MTS assay according tothe manufacturer's protocol. Absorbance values for the formazon dye aremeasured using an automated plate reader (Molecular Dynamics, Sunnyvale,Calif.). IC₅₀ values are calculated from the data using GRAPHPAD™software (GraphPad Software, Inc., San Diego, Calif.). Each experimentis performed in triplicate.

The cytotoxicity of the candidate modified aptamers is also evaluatedwith regard to prostate cells that have been obtained from normalprostate tissue derived from radical prostatectomies. A procedure forestablishing primary human epithelial cell cultures from thedisease-free regions of the tissue (based on histology) has beendescribed (Peehl et al., In Vitro Cell Dev. Biol. 22: 90 (1986); Peehlet al., In Vitro Cell Dev. Biol. 24: 530-536 (1988); Barreto et al.,Cancer Epidemiol. Biomarkers Prev. 9: 265-270 (2000). The cultures aregrown in MCDB 105 medium (Sigma, St. Louis, Mo.) that has beensupplemented with growth factors and hormones as described previously(Peehl et al., In Vitro Cell Dev Biol 22: 90 (1986)). Trypan blueexclusion is used as a marker of viable cells. The data are analyzed toidentify those candidate modified aptamers sequences that are highlyefficient at killing PC cells that express xPSM, but that are notcytotoxic towards normal prostate cells. Candidate modified aptamersthat display the greatest differential cytotoxicity are furtherdeveloped as drugs for the treatment of human PC.

Results from a NCI 60 cell line screen show that FdUMP[10] is cytotoxictowards prostate cancer cells at physiologically relevant drugconcentrations (10 nM-100 nM). Further, FdUMP[10] was found to besubstantially more cytotoxic towards prostate cancer cells than either5FU or FdU which are potential degradatory products of FdUMP[10]. MTSassays and clonogenic assays also show that FdUMP[10] is substantiallymore cytotoxic towards DU145 and PC3 prostate cancer cells than anymonomeric fluoropyrimidine (5FU, FdU, FdUMP). These data are consistentwith FdUMP[10] being able to penetrate prostate cancer cells in intactform, without prior degradation to monomeric fluoropyrimidine products.

The cytotoxicity toward LNCaP prostrate cancer cells by a modified RNAaptamer containing a FdUMP[9] tail as compared with the unmodifiedparent RNA aptamer is illustrated in FIGS. 6A and 6B. As shown in FIGS.6A and 6B, the modification of the parent aptamer with FdUMP[9]increases the cytotoxicity toward the target prostate cancer cells.

FIGS. 7A and 7B further illustrate the cytotoxicity of the modifiedaptamers as assessed using the MTS assays. Since fluorescence microscopystudies indicated that substantial uptake of the modified aptameroccurred within 45 min in targeted LNCaP cells, the LNCaP cells (A) andthe PC3 cells (B) were exposed to the modified aptamer for 40 min.Following exposure to the modified aptamer, the medium containing themodified aptamer was removed and replaced with fresh medium. The cellswere then incubated for 48 hours. The 48 hours in drug-free mediumallows cells to metabolize the modified aptamer and progress through thecell-cycle. Cell viability was measured using the MTS assay. Resultsshow that the LNCaP cells express the PSMA target for the aptamer anddisplay dose-dependent reduction in percent viable cells (FIG. 7A). ThePC3 cells are less sensitive to the modified aptamer with nocytotoxicity at the lower concentrations (FIG. 7B).

To determine if extracellular degradation is important for thecytotoxicity of FdUMP[10] towards prostate cancer cells, cells wereincubated with either an inhibitor of phosphatase activity or aninhibitor of nucleotidase activity. Neither inhibitor substantiallyreduced the cytotoxicity of FdUMP[10] towards PC cells, indicating thatprostate cancer cells efficiently take up FdUMP[10] in multimeric form.Other types of cancer cells (e.g. colon cancer) were less efficient inuptake of FdUMP[10] multimer than were prostate cancer cells. Thesestudies demonstrate that ODNs can be prepared which are selectivelytaken up by certain types of cancer cells. Studies have shown that ODNscan be taken up into cells by an endocytotic mechanism (Loke et al.,Proc. Natl. Acad. Sci. USA 86: 3474-3478 (1989)).

In addition, cytotoxicity has been assessed in LNCaP cells and PC3 cellsusing the MTS assay following a 1 hour exposure to several modifiedaptamers. After the one hour exposure the medium containing the drug wasremoved and replaced with fresh medium. The cells were then incubatedfor 48 h. Cell viability was measured using an MTS assay. The resultsfrom a typical experiment are shown in FIG. 8. The results show that theA10-3:T[10] aptamer does not reduce the viability of either LNCaP or PC3cells over the concentration range evaluated (1.0×10⁻⁶-6.25×10⁻⁸ M).Thus, modified aptamers are not inherently cytotoxic to either LNCaP orPC3 cells. In contrast, the A10-3:FdU[9]dC aptamer decreased theviability of both LNCaP and PC3 cells in a concentration-dependentmanner. A10-3:FdU[9]dC decreased the viability of LNCaP cells to agreater extent than PC3 at all concentrations evaluated for a one hourdrug exposure. The reduction in cell viability was nearly equal forlonger exposure times. These data are consistent with the reduction incell viability occurring from a combination of two processes: 1)Selective uptake of A10-3:FdU[9]dC into LNCaP cells via an endocytoticprocess; and 2) Extracellular degradation of A10-3:FdU[9]dC to releasemonomeric FdU. These results suggest that modifying the rate ofextracellular degradation of modified aptamers may provide additionalselectivity for targeted cancer cells.

The results also demonstrate the high degree of potency ofA10-3:FdU[9]dC to PC cells. A one hour exposure to 1×10⁻⁶ MA10-3:FdU[9]dC reduced the viability of PC3 cells by nearly 50% andreduced the viability of LNCaP cells to an even greater extent. Longerexposure times resulted in nearly complete loss of cell viability. Thesedata are in sharp contrast to the effects of 5FU towards these cells.Studies from our laboratory and from the NCI 60 cell line screen haveshown that FdUMP[10], a linear homopolymer often FdUMP nucleotides, is1,585 time more potent than 5FU towards DU145 PC cells and 631 timesmore potent than 5FU towards PC3 cells. The IC₅₀ of FdUMP[10] towardsDU145 cells is 5.01×10⁻⁹ M (GI₅₀ value from the NCI 60 cell line screen)and both PC3 and LNCaP cells also are highly sensitive to FdUMP[10] (butnot 5FU).

The mechanistic basis for the sensitivity of PC cells to FdUMP[10] (butnot 5FU) is currently under investigation. PC cells may be highlysensitive to activated fluoropyrimidines (FPs; e.g. FdUMP and FdUTP)because FdU-substituted DNA acts as a topoisomerase 1 (Top 1) poison. PCcells express relatively high levels of Top 1 and are sensitive to otherTop 1 poisons, such as the camptothecins (CPTs). CPTs have not shownefficacy for treatment of PC as a result of dose-limiting toxicities.5FU is not efficiently metabolized to FdUMP and FdUTP in PC cells andhence 5FU, and pro-drugs of 5FU (e.g. capecitabine) are not useful forthe treatment of PC. FdUMP[10] is very well-tolerated in vivo, andtrypan blue exclusion assays show a large differential sensitivitybetween PC cells and normal prostatic epithelial cells. Modifiedaptamers are expected to have greater intrinsic stability in plasmarelative to FdUMP[10] because they are highly structured. Thus, modifiedaptamers are expected to be even better tolerated in vivo thanFdUMP[10], and also to display greater selective cytotoxicity fortargeted PC cells than FdUMP[10].

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of generating a nucleic acid of interest, which nucleic acidspecifically binds to an extracellular surface protein expressed by acell of interest, and which nucleic acid is capable of beinginternalized by said cell of interest, said method comprising the stepsof: combining a first pool comprising different nucleic acids with saidextracellular surface protein; selecting a first subpopulation ofnucleic acids from said first pool, said first subpopulation comprisingat least one nucleic acid that specifically binds to said extracellularsurface protein; amplifying said at least one nucleic acid of said firstsubpopulation; and selecting a second subpopulation comprising at leastone nucleic acid species from said first subpopulation which isinternalized by said cell of interest.
 2. The method of claim 1, whereinsaid cell of interest is a cancer cell.
 3. The method of claim 1,wherein said cell of interest is a microbial cell.
 4. The method ofclaim 1, wherein said cell of interest is a parasitic cell.
 5. Themethod of claim 1, wherein said nucleic acid comprises a compound ofinterest.
 6. The method of claim 5, wherein said compound of interest isan active compound.
 7. The method of claim 5, wherein said compound ofinterest is a detectable group.
 8. The method of claim 5, where saidnucleic acid comprises more than one compound of interest.
 9. The methodof claim 6, wherein said active compound comprises cytotoxicnucleotides.
 10. The method of claim 9, wherein said cytotoxicnucleotides comprise poly-FdUMP.
 11. The method of claim 10, whereinsaid poly-FdUMP comprises FdUMP[9] or FdUMP[10].
 12. The method of claim1, wherein said amplifying step further comprises sequencing at leastone selected nucleic acid from said first subpopulation.
 13. The methodof claim 1, further comprising the step of synthesizing a syntheticnucleic acid having a sequence corresponding to said selected nucleicacid, and further comprising a compound of interest.
 14. The method ofclaim 1, wherein said extracellular surface protein is an extracellularsurface protein differentially expressed by cancer cells.
 15. The methodof claim 14, wherein said extracellular surface protein comprises anextracellular surface portion of prostate specific membrane antigen(PSMA).
 16. The method of claim 1, wherein said first pool comprisesnucleic acids having a predetermined bias.
 17. A method of generating anucleic acid of interest, which nucleic acid specifically binds to anextracellular surface protein expressed by a cell of interest, whichnucleic acid is capable of being internalized by said cell of interest,and which nucleic acid comprises one or more compounds of interest to bedelivered to said cell of interest, said method comprising the steps of:combining a first pool comprising different nucleic acids with saidextracellular surface protein; selecting a first subpopulation ofnucleic acids from said first pool, said first subpopulation comprisingat least one nucleic acid that specifically binds to said extracellularsurface protein; amplifying said at least one nucleic acid of said firstsubpopulation; selecting a second subpopulation comprising at least onenucleic acid species from said first subpopulation which is internalizedby said cell of interest; determining a first chemical structure of anucleic acid from said second population; determining a second chemicalstructure of a nucleic acid which is an analog of said nucleic acid fromsaid second population, said analog comprising one or more of saidcompounds of interest; analyzing the folding properties of said firstchemical structure; analyzing the folding properties of said secondchemical structure; comparing the folding properties of said firstchemical structure with that of said second chemical structure; andgenerating a nucleic acid of interest based upon said comparing.
 18. Themethod of claim 17, wherein said cell of interest is a cancer cell. 19.The method of claim 17, wherein said cell of interest is a microbialcell.
 20. The method of claim 17, wherein said cell of interest is aparasitic cell.
 21. The method of claim 17, wherein said compound ofinterest is an active compound.
 22. The method of claim 17, wherein saidcompound of interest is a detectable group.
 23. The method of claim 17,where said nucleic acid of interest comprises more than one compound ofinterest.
 24. The method of claim 21, wherein said active compoundcomprises cytotoxic nucleotides.
 25. The method of claim 24, whereinsaid cytotoxic nucleotides comprise poly-FdUMP.
 26. The method of claim25, wherein said poly-FdUMP comprises FdUMP[9] or FdUMP[10].
 27. Themethod of claim 17, wherein said amplifying step further comprisessequencing at least one selected nucleic acid from said firstsubpopulation.
 28. The method of claim 17, wherein said determining afirst chemical structure comprises sequencing said at least one selectednucleic acid from said second subpopulation.
 29. The method of claim 17,wherein said generating step comprises synthesizing a synthetic nucleicacid having a sequence corresponding to said selected nucleic acid ofinterest.
 30. The method of claim 17, wherein said extracellular surfaceprotein is an extracellular surface protein differentially expressed bycancer cells.
 31. The method of claim 30, wherein said extracellularsurface protein comprises an extracellular surface portion of prostatespecific membrane antigen (PSMA).
 32. The method of claim 17, whereinsaid first pool comprises nucleic acids having a predetermined bias.